The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and B15% of their Z-disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed-and barbed-end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z-disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.

Background-The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results-SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (PϽ0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (nϭ7), intermediate in those with pseudonormal filling (nϭ6), and lowest in the group with restrictive filling (nϭ7). Mechanical measurements on LV muscle strips dissected from these hearts (nϭ8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O 2 consumption (nϭ10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Conclusions-Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.

Abstract-Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is Ϸ0.05 in mouse and Ϸ1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implications of our findings to cardiac function are discussed. (Circ Res. 2000;86:59-67.)Key Words: compliance Ⅲ passive tension Ⅲ diastolic force Ⅲ mechanical properties Ⅲ myocyte Ⅲ connectin D uring diastole, the myocardium stretches and passive force (F) is generated. A major contributor to this force is the giant protein titin, spanning the half-sarcomere from the Z-band to the M-line. When sarcomere length (SL) increases during diastole, the I-band region of titin extends and force develops. The shape of F-SL relation of titin is expected to influence ventricular filling during diastole and ventricular emptying during systole. In addition to influencing ventricular filling, titin also helps to maintain the structural integrity of the contracting sarcomere (for reviews, see References 1-5).The force of titin arises from its extensible I-band region, which consists of 2 main segment types: (1) a segment rich in proline (P), glutamate (E), valine (V), and lysine (K) residues (the so-called PEVK segment) and (2) serially linked immunoglobulin (Ig)-like domains (tandem Ig segments) flanking this PEVK segment. 6 Several titin isoforms are now known, all of which contain PEVK and tandem Ig segments. In addition to these common segments, the extensible region of the N2B isoform contains the N2B element (3 Ig domains and a 572-residue unique sequence),...